The vast majority of assays performed on mitochondrial function are on large quantities of mitochondria, typically requiring 107 cells worth of sample. While there exist a few techniques such as flow cytometry and capillary electrophoresis that can be used to investigate individual mitochondria, none of these are suitable for the studies and application envisioned with the technology described herein.
Several studies have demonstrated the application of flow cytometry to the analysis of individual mitochondria and provide evidence for a heterogeneous population, thus further motivating the technology development presented herein. Flow cytometry has the advantage of using existing commercially available instruments. Flow cytometry provides a “snap shot” of a single mitochondrial state (e.g. JC-1 fluorescence, forward scatter and side scatter). In this way, statistical analysis of mitochondria under various conditions and states can be obtained. The use of flow cytometry cannot be used to track the status of individual mitochondria over a longer period of time. In flow cytometry, a single mitochondria flows quickly past the detectors for a short interrogation time (less than 1 ms), never to be interrogated again.
Similar to flow cytometry, capillary electrophoresis with laser-induced fluorescence detection allows for analysis of single mitochondria. In this technique, a 50 μm capillary guides individual mitochondria which migrate in response to a high electric field (ca. 200 V/cm). Using this technique, a variety of mitochondrial properties can be assayed, such as the electrophoretic mobility, the cardiolipin content, and ROS production. As in flow cytometry, single mitochondria are analyzed at a snapshot in time as they migrate passed the detection window. Again, the technique is able to analyze large number of mitochondria and their variations. However, an extended duration analysis of a single mitochondrion is not possible. In addition, a membrane potential assay has not yet been demonstrated with this approach. Possible complications of the large electric field to the membrane potential have not yet been addressed.
An alternative technique to immobilize isolated mitochondria involves adhesion to a glass microscope slide. This technique allows visualization and characterization of up to hundreds of individual mitochondria in a single field of view. A disadvantage of this approach, however, is the large fluorescence background of the fluorophore outside of the mitochondria. If one seeks to quantitatively determine Δψm using potential sensitive dyes, it is necessary to carefully measure the ratio of the dye fluorescence intensity at the inside to the outside of the mitochondria. The measurement is complicated by the large diffuse fluorescence background of the fluorophore outside of the mitochondria. In addition, parallel processing of multiple analytes is not possible on a glass slide.